Combined derivatization and high-performance liquid chromatography with fluorescence and ultraviolet detection for simultaneous analysis of octreotide and gabexate mesylate metabolite in human pancreatic juice samples.

A simple and sensitive method based on the combination of derivatization and high-performance liquid chromatography with ultraviolet and fluorimetric detection was developed for the simultaneous determination of octreotide and gabexate mesylate metabolite in human pancreatic juice samples. Parameters of the derivatization procedure affecting extraction efficiency were optimized. The developed method was validated according to the International Conference on Harmonization guidelines. The calibration curves were linear over a range of 0.1-15 µg/mL for octreotide and 0.20-15 µg/mL for gabexate mesylate metabolite. Derivatized products of octreotide and gabexate mesylate metabolite were separated on a Luna C18 column (4.6 × 250 mm; 5 µm particle size) using a gradient with a run time of 36 min, without further purification. The limits of detection were 0.025 and 0.05, respectively, for octreotide and gabexate mesylate metabolite. This paper reports the validation of a quantitative high performance liquid chromatography-photodiode array-fluorescence (HPLC-PDA-FL) method for the simultaneous analysis of octreotide and gabexate mesylate metabolite in pancreatic juice by protein precipitation using zinc sulfate-methanol-acetonitrile containing the derivatizing reagent, 4-fluoro-7-nitro-[2,1,3]-benzoxadiazole (NBD-F). Derivatized products of octreotide and gabexate mesylate metabolite were separated on a Luna C18 column (4.6 × 250 mm; 5 µm particle size) using a gradient with a run time of 36 min, without further purification. The method was validated over the concentration ranges 0.1-15 and 0.2-15 µg/mL for octreotide and gabexate mesylate metabolite, respectively, in human pancreatic juice. Biphalin and methyl-p-hydroxybenzoate were used as the internal standards. This method was successfully utilized to support clinical studies in humans. The results from assay validations show that the method is selective, sensitive and robust. The limit of quantification of the method was 0.1 µg/mL for octreotide and 0.2 µg/mL for gabexate mesylate metabolite, and matrix matched standard curves showed a good linearity up to 15 µg/mL. In the entire analytical range the intra- and inter-day precision (RSD%) values were respectively ≤5.9% and ≤3.1% for octreotide and ≤2.0% and ≤3.9% for gabexate mesylate metabolite. For both analytes the intra- and inter-day accuracy (bias) values ranged respectively from -6.8 to -2.5% and from -4.6 to -5.7%. This method utilizes derivatization with NBD-F and provides adequate sensitivity for both drugs.

Matrix metalloproteinases 2, 3, 8, 9, and 13 in the peri-implant soft tissues around titanium and zirconium oxide healing caps.

PURPOSE: This human study sought to compare, from an immunohistochemical point of view, matrix metalloproteinases (MMPs) 2, 3, 8, 9, and 13 in the soft tissues around titanium and zirconium oxide healing caps. MATERIALS AND METHODS: Five patients participated in this study. All patients received 3.8 × 11-mm dental implants, which were left to heal in a nonsubmerged (single-stage) mode. Healing caps (3.8 mm in diameter and 3.0 mm in height) were inserted in all implants. Half of the implants were randomly supplied with standard, prefabricated caps of commercially pure titanium (control), while the other half were randomly provided with zirconium oxide caps (test). After a 6-month healing period, gingival biopsy specimens were obtained with a circular scalpel around the healing caps of both groups, without unscrewing or removing the healing caps, and the samples underwent immunohistochemical processing for MMPs 2, 3, 8, 9, and 13. RESULTS: Statistically significant differences were found in the values of MMP-8 in the cells of the inflammatory infiltrate, with higher values for the titanium samples. Statistically significantly higher values were found, also in the titanium samples, for MMP-9 in the endothelial cells of the blood vessels. No statistically significant differences were found for any other MMPs. CONCLUSIONS: The present results showed that the soft tissues around titanium healing caps underwent a higher rate of restorative processes, most probably correlated to the MMP levels observed in the tissues.

Cathepsins and pancreatic cancer: the 2012 update.

Pancreatic cancer is the result of distinctive genetic and epigenetic disturbances. This multistep process is in part well-defined and includes alterations in oncogenes and suppressor genes that control proliferation, apoptosis, angiogenesis, invasion and cell migration. Cathepsins are proteolytic enzymes and represent potential therapeutic targets in human tumors. Cathepsins predominantly function as endopeptidases within endolysosomal vesicles of normal cells and they are involved in physiological processes such as protein turnover, differentiation and apoptosis. In various types of malignancies, cathepsins have been associated with tumor progression and metastasis. Growing evidence and direct proofs suggest that cathepsins are highly up-regulated in pancreatic cancer and contribute to the development and progression of the cancer phenotype. In this review, the role of cathepsins in pancreatic cancer tumorigenesis is reported and discussed. Some critical aspects will be underlined such as specificity of cathepsin activity in pancreatic cancer and in its precursor lesions; the genetic perturbation and the intracellular signaling pathway activated by cathepsins as reported in preclinical models and in human tissues; the preliminary results and the oncological effects of cathepsin inhibitors currently tested on pancreatic cancer cells; the role of combined therapy based on chemotherapeutic agents and cathepsin inhibition. Although mounting evidences indicate that cysteine cathepsins are potential therapeutic targets in pancreatic cancer, as suggested by their functional role in controlling invasiveness and metastasis, it remains to be seen whether the promising benefits of pharmacological inhibitors observed in preclinical study might be translated to the current clinical practice.

Targeting phosphoinositide 3-kinase pathways in pancreatic cancer--from molecular signalling to clinical trials.

Pancreatic cancer has one of the poorest prognoses among all cancers partly because of its silent nature and tendency for late discovery but also because of its persistent resistance to chemotherapy. At present there are very limited treatment alternatives for pancreatic cancer, hence the need to develop novel and more efficient drugs. It is well known that mutations in K-Ras oncogene accumulate early in the disease progression and occur in almost all of pancreatic ductal adenocarcinomas (PDAC). A key downstream target of the Ras family is phosphoinositide 3-kinase (PI3K), the enzyme responsible for generation of 3-phosphorylated phosphoinositides and activation of Akt (Protein Kinase B/Akt). The PI3K/Akt pathway is involved in inhibition of apoptosis and stimulation of cell proliferation and it is has been estimated that at least 50% of all cancer types are related to deregulation of this signalling pathway. In this review we will discuss how the PI3K/Akt/mTOR signaling network is altered in pancreatic cancer and further give an overview of preclinical and clinical studies where this pathway has been targeted.